Chronic lymphocytic leukemia cell line

ABSTRACT

A CLL line, CLL-AAT, and the preparation and characterization of antibodies using said cell line is disclosed.

RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No.60/254,113 filed Dec. 8, 2000, the disclosure of which is incorporatedherein by reference.

TECHNICAL FIELD

Cell lines derived from chronic lymphocytic leukemia (CLL) cells and theuses thereof in the study and treatment of CLL disease are disclosed. Inparticular, this disclosure relates to a CLL cell line designated“CLL-AAT”, deposited on Nov. 28, 2001 with the American Type CultureCollection (Manassas, Va., USA) in accordance with the terms of theBudapest Treaty under ATCC accession no. ______.

BACKGROUND

Chronic Lymphocytic Leukemia (CLL) is a disease of the white blood cellsand is the most common form of leukemia in the Western Hemisphere. CLLrepresents a diverse group of diseases relating to the growth ofmalignant lymphocytes that grow slowly but have an extended life span.CLL is classified in various categories that include, for example,B-cell chronic lymphocytic leukemia (B-CLL) of classical and mixedtypes, B-cell and T-cell prolymphocyic leukemia, hairy cell leukemia,and large granular lymphocytic leukemia.

Of all the different types of CLL, B-CLL accounts for approximately 30percent of all leukemias. Although it occurs more frequently inindividuals over 50 years of age it is increasingly seen in youngerpeople. B-CLL is characterized by accumulation of B-lymphocytes that aremorphologically normal but biologically immature, leading to a loss offunction. Lymphocytes normally function to fight infection. In B-CLL,however, lymphocytes accumulate in the blood and bone marrow and causeswelling of the lymph nodes. The production of normal bone marrow andblood cells is reduced and patients often experience severe anemia aswell as low platelet counts. This can pose the risk of life-threateningbleeding and the development of serious infections because of reducednumbers of white blood cells.

To further understand diseases such as leukemia it is important to havesuitable cell lines that can be used as tools for research on theiretiology, pathogenesis and biology. Examples of malignant humanB-lymphoid cell lines include pre-B acute lymphoblasticleukemia (Reh),diffuse large cell lymphoma (WSU-DLCL2), and Waldenstrom'smacroglobulinemia (WSU-WM). Unfortunately, many of the existing celllines do not represent the clinically most common types of leukemia andlymphoma.

The use of Epstein Barr Virus (EBV) infection in vitro has resulted insome CLL derived cell lines, in particular B-CLL cells lines, that arerepresentative of the malignant cells. The phenotype of these cell linesis different than that of the in vivo tumors and instead the features ofB-CLL lines tend to be similar to those of Lymphoblastoid cell lines.Attempts to immortalize B-CLL cells with the aid of EBV infection havehad little success. The reasons for this are unclear but it is knownthat it is not due a lack of EBV receptor expression, binding or uptake.Wells et al. found that B-CLL cells were arrested in the G1/S phase ofthe cell cycle and that transformation associated EBV DNA was notexpressed. This suggests that the interaction of EBV with B-CLL cells isdifferent from that with normal B cells. EBV-transformed CLL cell linesmoreover appear to differentiate, possessing a morphology more similarto lymphoblastoid cell lines (LCL) immortalized by EBV.

An EBV-negative CLL cell line, WSU-CLL, has been established previously(Mohammad et al., (1996) Leukemia 10(1):130-7). However, no other suchcell lines are known.

There remains a need in the art, therefore, for a CLL cell line whichhas not been established by transformation with EBV, and which expressessurface markers characteristic of primary CLL cells.

SUMMARY

In one embodiment an CLL cell line of malignant origin is provided thatis not established by immortalisation with EBV. The cell line, which wasderived from primary CLL cells, and is deposited under ATCC accessionno. ______. In a preferred embodiment, the cell line is CLL-AAT. CLL-MTis B-CLL cell line, derived from a B-CLL primary cell.

In a further aspect, the CLL-AAT cell line is used to generatemonoclonal antibodies useful in the diagnosis and/or treatment of CLL.Antibodies may be generated by using the cells as disclosed herein asimmunogens, thus raising an immune response in animals from whichmonoclonal antibodies may be isolated. The sequence of such antibodiesmay be determined and the antibodies or variants thereof produced byrecombinant techniques. In this aspect, “variants” includes chimeric,CDR-grafted, humanized and fully human antibodies based on the sequenceof the monoclonal antibodies.

Moreover, antibodies derived from recombinant libraries (“phageantibodies”) may be selected using the cells described herein, orpolypeptides derived therefrom, as bait to isolate the antibodies on thebasis of target specificity.

In a still further aspect, antibodies may be generated by panningantibody libraries using primary CLL cells, or antigens derivedtherefrom, and further screened and/or characterized using the cell lineof the invention. Accordingly, a method for characterizing an antibodyspecific for CLL is provided, which includes assessing the binding ofthe antibody to a CLL cell line.

In a further aspect, there is provided a method for identifying proteinsuniquely expressed in CLL cells employing the CLL-MT cell line, bymethods well known to those, skilled with art, such as byimmunoprecipitation followed by mass spectroscopy analyses. Suchproteins may be uniquely expressed in the CLL-AAT cell line, or inprimary cells derived from CLL patients.

Small molecule libraries (many available commercially) may be screenedusing the CLL-AAT cell line in a cell-based assay to identify agentscapable of modulating the growth characteristics of the cells. Forexample, the agents may be identified which modulate apoptosis in theCLL-MT cell line, or which inhibit growth and/or proliferation thereof.Such agents are candidates for the development of therapeutic compounds.

Nucleic acids isolated from CLL-MT cell lines may be used in subtractivehybridization experiments to identify CLL-specific genes or in microarray analyses (e.g., gene chip experiments). Genes whose transcriptionis modulated in CLL cells may be identified. Polypeptide or nucleic acidgene products identified in this manner are useful as leads for thedevelopment of antibody or small molecule therapies for CLL.

In a preferred aspect, the CLL-AAT cell line may be used to identifyinternalizing antibodies, which bind to cell surface components whichare internalized by the cell. Such antibodies are candidates fortherapeutic use. In particular, single-chain antibodies, which remainstable in the cytoplasm and which retain intracellular binding activity,may be screened in this manner.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. schematically illustrates typical steps involved in cell surfacepanning of antibody libraries by magnetically-activated cell sorting(MACS).

FIG. 2. is a graph showing the results of whole cell ELISA demonstratingbinding of selected scFv clones to primary B-CLL cells and absence ofbinding to normal human PBMC. The designation 2°+3° in this and otherfigures refers to negative control wells stained with Mouse Anti-HA anddetecting antimouse antibodies alone. The designation RSC-S Library inthis and other figures refers to soluble antibodies prepared fromoriginal rabbit scFv unpanned library. The designation R3/RSC-S Pool inthis and other figures refers to soluble antibodies prepared from entirepool of scFv antibodies from round 3 of panning. Anti-CD5 antibody wasused as a positive control to verify that equal numbers of B-CLL andPBMC cells were plated in each well.

FIG. 3. is a graph showing the results of whole cell ELISA comparingbinding of selected scfv antibodies to primary B-CLL cells and normalprimary human B cells. Anti-CD19 antibody was used as a positive controlto verify that equal numbers of B-CLL and normal B cells were plated ineach well. Other controls were as described in the legend to FIG. 2.

FIG. 4. is a graph showing the results of whole cell ELISA used todetermine if scFv clones bind to patient-specific (i.e. idiotype) orblood type-specific (i.e. HLA) antigens. Each clone was tested forbinding to PBMC isolated from 3 different B-CLL patients. Clones thatbound to 1 patient sample were considered to be patient or bloodtype-specific.

FIG. 5. is a graph showing the results of whole cell ELISA comparingbinding of scFv clones to primary B-CLL cells and three human leukemiccell lines. Ramos is a mature B cell line derived from a Burkitt'slymphoma. RL is a mature B cell line derived from a non-Hodgkin'slymphoma. TF-I is an erythroblastoid cell line derived from aerythroleukemia.

FIG. 6. is a graph showing the results of whole cell ELISA comparingbinding of scFv clones to primary B-CLL cells and CLL-AAT, a cell linederived from a B-CLL patient. TF-I cells were included as a negativecontrol.

FIG. 7 shows the binding specificity of scFv antibodies in accordancewith this disclosure as analyzed by 3-color flow cytometry. In normalperipheral blood mononuclear cells, the antigen recognized by scFv-9 ismoderately expressed on B lymphocytes and weakly expressed on asubpopulation of T lymphocytes. PBMC from a normal donor were analyzedby 3-color flow cytometry using anti-CD5-FITC, anti-CD19-PerCP, andscFv-9/Anti-HA-biotin/streptavidin-PE.

FIG. 8 shows the expression levels of antigens recognized by scFvantibodies in accordance with this disclosure.

FIG. 9 is Table 1 which provided a summary of CDR sequences and bindingspecificities of selected scFv antibodies.

FIG. 10. is Table 2 which shows a summary of flow cytometry resultscomparing expression levels of scFv antigens on primary CLL cells vs.normal PBMC as described in FIG. 8.

DETAILED DESCRIPTION

Definitions

“CLL”, as used herein, refers to chronic lymphocytic leukemia involvingany lymphocyte, including but not limited to various developmentalstages of B cells and T cells, including but not limited to B cell CLL.B-CLL, as used herein, refers to leukemia with a mature B cell phenotypewhich is CD5⁺, CD23⁺, CD20^(dim+), sIg^(dim+) and arrested in G0/G1 ofthe cell cycle.

“Malignant origin” refers to the derivation of the cell line frommalignant CLL primary cells, as opposed to non-proliferating cells whichare transformed, for example, with EBV. Cell lines according to thisdisclosure may be themselves malignant in phenotype, or not. A CLL cellhaving a “malignant” phenotype encompasses cell growth unattached fromsubstrate media characterized by repeated cycles of cell growth andexhibits resistance to apoptosis.

Preparation of Cell Lines

Cell lines may be produced according to established methodologies knownto those skilled in the art. In general, cell lines are produced byculturing primary cells derived from a patient until immortalized cellsare spontaneously generated in culture. These cells are then isolatedand further cultured, to produce clonal cell populations or cellsexhibiting resistance to apoptosis.

For example, CLL cells may be isolated from peripheral blood drawn froma patient suffering from CLL. The cells may be washed, and optionallyimmunotyped in order to determine the type(s) of cells present.Subsequently, the cells may be cultured in a medium, such as a mediumcontaining IL-4. Advantageously, all or part of the medium is replacedone or more times during the culture process. Cell lines may be isolatedthereby, and will be identified by increased growth in culture.

Preparation of Monoclonal Antibodies

Antibodies, as used herein, refers to complete antibodies or antibodyfragments capable of binding to a selected target. Included are Fv,ScFv, Fab′ and F(ab′)₂, monoclonal and polyclonal antibodies, engineeredantibodies (including chimeric, CDR-grafted and humanized, fully humanantibodies, and artificially selected antibodies), and synthetic or semisynthetic antibodies produced using phage display or alternativetechniques. Small fragments, such Fv and ScFv, possess advantageousproperties for diagnostic and therapeutic applications on account oftheir small size and consequent superior tissue distribution.

The antibodies are especially indicated for diagnostic and therapeuticapplications. Accordingly, they may be altered antibodies comprising aneffector protein such as a toxin or a label. Especially preferred arelabels which allow the imaging of the distribution of the antibody invivo. Such labels may be radioactive labels or radiopaque labels, suchas metal particles, which are readily visualisable within the body of apatient. Moreover, the labels may be fluorescent labels or other labelswhich are visualisable on tissue samples removed from patients.

Recombinant DNA technology may be used to improve the antibodiesproduced in accordance with this disclosure. Thus, chimeric antibodiesmay be constructed in order to decrease the immunogenicity thereof indiagnostic or therapeutic applications. Moreover, immunogenicity may beminimized by humanizing the antibodies by CDR grafting and, optionally,framework modification. See, U.S. Pat. No. 5,225,539, the contents ofwhich are incorporated herein by reference.

Antibodies may be obtained from animal serum, or, in the case ofmonoclonal antibodies or fragments thereof produced in cell culture.Recombinant DNA technology may be used to produce the antibodiesaccording to established procedure, in bacterial or preferably mammaliancell culture. The selected cell culture system preferably secretes theantibody product.

In another embodiment, a process for the production of an antibodydisclosed herein includes culturing a host, e.g. E. coli or a mammaliancell, which has been transformed with a hybrid vector. The vectorincludes one or more expression cassettes containing a promoter operablylinked to a first DNA sequence encoding a signal peptide linked in theproper reading frame to a second DNA sequence encoding the antibodyprotein. The antibody protein is then collected and isolated.Optionally, the expression cassette may include a promoter operablylinked to polycistronic, for example bicistronic, DNA sequences encodingantibody proteins each individually operably linked to a signal peptidein the proper reading frame.

Multiplication of hybridoma cells or mammalian host cells in vitro iscarried out in suitable culture media, which include the customarystandard culture media (such as, for example Dulbecco's Modified EagleMedium (DMEM) or RPMI 1640 medium), optionally replenished by amammalian serum (e.g. fetal calf serum), or trace elements and growthsustaining supplements (e.g. feeder cells such as normal mouseperitoneal exudate cells, spleen cells, bone marrow macrophages,2-aminoethanol, insulin, transferrin, low density lipoprotein, oleicacid, or the like). Multiplication of host cells which are bacterialcells or yeast cells is likewise carried out in suitable culture mediaknown in the art. For example, for bacteria suitable culture mediainclude medium LE, NZCYM, NZYM, NZM, Terrific Broth, SOB, SOC, 2×YT, orM9 Minimal Medium. For yeast, suitable culture media include medium YPD,YEPD, Minimal Medium, or Complete Minimal Dropout Medium.

In vitro production provides relatively pure antibody preparations andallows scale-up to give large amounts of the desired antibodies.Techniques for bacterial cell, yeast, plant, or mammalian cellcultivation are known in the art and include homogeneous suspensionculture (e.g. in an airlift reactor or in a continuous stirrer reactor),and immobilized or entrapped cell culture (e.g. in hollow fibres,microcapsules, on agarose microbeads or ceramic cartridges).

Large quantities of the desired antibodies can also be obtained bymultiplying mammalian cells in vivo. For this purpose, hybridoma cellsproducing the desired antibodies are injected into histocompatiblemammals to cause growth of antibody-producing tumors. Optionally, theanimals are primed with a hydrocarbon, especially mineral oils such aspristane (tetramethyl-pentadecane), prior to the injection. After one tothree weeks, the antibodies are isolated from the body fluids of thosemammals. For example, hybridoma cells obtained by fusion of suitablemyeloma cells with antibody-producing spleen cells from Balb/c mice, ortransfected cells derived from hybridoma cell line Sp2/0 that producethe desired antibodies are injected intraperitoneally into Balb/c miceoptionally pre-treated with pristine. After one to two weeks, asciticfluid is taken from the animals.

The foregoing, and other, techniques are discussed in, for example,Kohler and Milstein, (1975) Nature 256:495-497; U.S. Pat. No. 4,376,110;Harlow and Lane, Antibodies: a Laboratory Manual, (1988) Cold SpringHarbor, the disclosures of which are all incorporated herein byreference. Techniques for the preparation of recombinant antibodymolecules is described in the above references and also in, for exampleWO97/08320; U.S. Pat. No. 5,427,908; U.S. Pat. No. 5,508,717; Smith,1985, Science, Vol. 225, pp 1315-1317; Parmley and Smith 1988, Gene 73,pp 305-318; De La Cruz et al, 1988, Journal of Biological Chemistry, 263pp 4318-4322; U.S. Pat. No. 5,403,484; U.S. Pat. No. 5,223,409;WO88/06630; WO92/15679; U.S. Pat. No. 5,780,279; U.S. Pat. No.5,571,698; U.S. Pat. No. 6,040,136; Davis et al., Cancer MetastasisRev., 1999;18(4):421-5; Taylor, et al., Nucleic Acids Research 20(1992): 6287-6295; Tomizuka et al., Proc. Nat. Academy of Sciences USA97(2) (2000): 722-727. The contents of all these references areincorporated herein by reference.

The cell culture supernatants are screened for the desired antibodies,preferentially by immunofluorescent staining of CLL cells, byimmunoblotting, by an enzyme immunoassay, e.g. a sandwich assay or adot-assay, or a radioimmunoassay.

For isolation of the antibodies, the immunoglobulins in the culturesupernatants or in the ascitic fluid may be concentrated, e.g. byprecipitation with ammonium sulfate, dialysis against hygroscopicmaterial such as polyethylene glycol, filtration through selectivemembranes, or the like. If necessary and/or desired, the antibodies arepurified by the customary chromatography methods, for example gelfiltration, ion-exchange chromatography, chromatography overDEAE-cellulose and/or (immuno-) affinity chromatography, e.g. affinitychromatography with a one or more surface polypeptides derived from aCLL cell line according to this disclosure, or with Protein-A or G.

Another embodiment provides a process for the preparation of a bacterialcell line secreting antibodies directed against the cell linecharacterized in that a suitable mammal, for example a rabbit, isimmunized with pooled CLL patient samples. A phage display libraryproduced from the immunized rabbit is constructed and panned for thedesired antibodies in accordance with methods well known in the art(such as, for example, the methods disclosed in the various referencesincorporated herein by reference).

Hybridoma cells secreting the monoclonal antibodies are alsocontemplated. The preferred hybridoma cells are genetically stable,secrete monoclonal antibodies described herein of the desiredspecificity and can be activated from deep-frozen cultures by thawingand recloning.

In another embodiment, a process is provided for the preparation of ahybridoma cell line secreting monoclonal antibodies directed to the CLLcell line is described herein. In that process, a suitable mammal, forexample a Balb/c mouse, is immunized with a one or more polypeptides orantigenic fragments thereof derived from a cell described in thisdisclosure, the cell line itself, or an antigenic carrier containing apurified polypeptide as described. Antibody-producing cells of theimmunized mammal are grown briefly in culture or fused with cells of asuitable myeloma cell line. The hybrid cells obtained in the fusion arecloned, and cell clones secreting the desired antibodies are selected.For example, spleen cells of Balb/c mice immunized with the cell line ofthe invention are fused with cells of the myeloma cell line PAI or themyeloma cell line Sp2/0-Ag 14, the obtained hybrid cells are screenedfor secretion of the desired antibodies, and positive hybridoma cellsare cloned.

Preferred is a process for the preparation of a hybridoma cell line,characterized in that Balb/c mice are immunized by injectingsubcutaneously and/or intraperitoneally between 10⁶ and 10⁷ cells of acell line in accordance with this disclosure several times, e.g. four tosix times, over several months, e.g. between two and four months. Spleencells from the immunized mice are taken two to four days after the lastinjection and fused with cells of the myeloma cell line PAI in thepresence of a fusion promoter, preferably polyethylene glycol.Preferably, the myeloma cells are fused with a three- to twenty-foldexcess of spleen cells from the immunized mice in a solution containingabout 30% to about 50% polyethylene glycol of a molecular weight around4000. After the fusion, the cells are expanded in suitable culture mediaas described hereinbefore, supplemented with a selection medium, forexample HAT medium, at regular intervals in order to prevent normalmyeloma cells from overgrowing the desired hybridoma cells.

In a further embodiment, recombinant DNA comprising an insert coding fora heavy chain variable domain and/or for a light chain variable domainof antibodies directed to the cell line described hereinbefore areproduced. The term DNA includes coding single stranded DNAs, doublestranded DNAs consisting of said coding DNAs and of complementary DNAsthereto, or these complementary (single stranded) DNAs themselves.

Furthermore, DNA encoding a heavy chain variable domain and/or a lightchain variable domain of antibodies directed to the cell line disclosedherein can be enzymatically or chemically synthesized DNA having theauthentic DNA sequence coding for a heavy chain variable domain and/orfor the light chain variable domain, or a mutant thereof. A mutant ofthe authentic DNA is a DNA encoding a heavy chain variable domain and/ora light chain variable domain of the above-mentioned antibodies in whichone or more amino acids are deleted or exchanged with one or more otheramino acids. Preferably said modification(s) are outside the CDRs of theheavy chain variable domain and/or of the light chain variable domain ofthe antibody in humanization and expression optimization applications.The term mutant DNA also embraces silent mutants wherein one or morenucleotides are replaced by other nucleotides with the new codons codingfor the same amino acid(s). The term mutant sequence also includes adegenerated sequence. Degenerated sequences are degenerated within themeaning of the genetic code in that an unlimited number of nucleotidesare replaced by other nucleotides without resulting in a change of theamino acid sequence originally encoded. Such degenerated sequences maybe useful due to their different restriction sites and/or frequency ofparticular codons which are preferred by the specific host, particularlyE. coli, to obtain an optimal expression of the heavy chain murinevariable domain and/or a light chain murine variable domain.

The term mutant is intended to include a DNA mutant obtained by in vitromutagenesis of the authentic DNA according to methods known in the art.

For the assembly of complete tetrameric immunoglobulin molecules and theexpression of chimeric antibodies, the recombinant DNA inserts codingfor heavy and light chain variable domains are fused with thecorresponding DNAs coding for heavy and light chain constant domains,then transferred into appropriate host cells, for example afterincorporation into hybrid vectors.

Recombinant DNAs including an insert coding for a heavy chain murinevariable domain of an antibody directed to the cell line disclosedherein fused to a human constant domain g, for example γ1, γ2, γ3 or γ4,preferably γ1 or γ4 are also provided. Recombinant DNAs including aninsert coding for a light chain murine variable domain of an antibodydirected to the cell line disclosed herein fused to a human constantdomain κ or λ, preferably κ are also provided.

Another embodiment pertains to recombinant DNAs coding for a recombinantpolypeptide wherein the heavy chain variable domain and the light chainvariable domain are linked by way of a spacer group, optionallycomprising a signal sequence facilitating the processing of the antibodyin the host cell and/or a DNA coding for a peptide facilitating thepurification of the antibody and/or a cleavage site and/or a peptidespacer and/or an effector molecule.

The DNA coding for an effector molecule is intended to be a DNA codingfor the effector molecules useful in diagnostic or therapeuticapplications. Thus, effector molecules which are toxins or enzymes,especially enzymes capable of catalyzing the activation of prodrugs, areparticularly indicated. The DNA encoding such an effector molecule hasthe sequence of a naturally occurring enzyme or toxin encoding DNA, or amutant thereof, and can be prepared by methods well known in the art.

Antibodies and antibody fragments disclosed herein are useful indiagnosis and therapy. Accordingly, a composition for therapy ordiagnosis comprising an antibody disclosed herein is provided.

In the case of a diagnostic composition, the antibody is preferablyprovided together with means for detecting the antibody, which may beenzymatic, fluorescent, radioisotopic or other means. The antibody andthe detection means may be provided for simultaneous, separate orsequential use, in a diagnostic kit intended for diagnosis.

Uses of the Cell Line of the Invention

There are many advantages to the development of a CLL cell line, as itprovides an important tool for the development of diagnostics andtreatments for CLL.

A cell line according to this disclosure may be used for in vitrostudies on the etiology, pathogenesis and biology of CLL. This assistsin the identification of suitable agents that are useful in the therapyof CLL disease.

The cell line may also be used to produce monoclonal antibodies for invitro and in vivo diagnosis of CLL, as referred to above, and for thescreening and/or characterization of antibodies produced by othermethods, such as by panning antibody libraries with primary cells and/orantigens derived from CLL patients.

The cell line may be used as such, or antigens may be derived therefrom.Advantageously, such antigens are cell-surface antigens specific forCLL. They may be isolated directly from cell lines according to thisdisclosure. Alternatively, a cDNA expression library made from a cellline described herein may be used to express CLL-specific antigens,useful for the selection and characterization of anti-CLL antibodies andthe identification of novel CLL-specific antigens.

Treatment of CLL using monoclonal antibody therapy has been proposed inthe art. Recently, Hainsworth (Oncologist 5 (5) (2000) 376-384) hasdescribed the current therapies derived from monoclonal antibodies.Lymphocytic leukemia in particular is considered to be a good candidatefor this therapeutic approach due to the presence of multiplelymphocyte-specific antigens on lymphocyte tumors.

Existing antibody therapies (such as Rituximab™, directed against theCD20-antigen, which is expressed on the surface of B-lymphocytes) havebeen used successfully against certain lymphocytic disease. However, alower density CD20 antigen is expressed on the surface of B-lymphocytesin CLL (Almasri et al., Am. J. Hematol., 40 (4) (1992) 259-263).

The CLL cell line described herein thus permits the development of novelanti-CLL antibodies having specificity for one or more antigenicdeterminants of the cell line of the invention, and their use in thetherapy and diagnosis of CLL.

In order that those skilled in the art may be better able to practicethe compositions and methods described herein, the following examplesare given for illustration purposes.

EXAMPLE 1

Isolation of Cell Line CLL-AAT

Establishment of the Cell Line

Peripheral blood from a patient diagnosed with CLL was obtained. The WBCcount was 1.6×10⁸/ml. Mononuclear cells were isolated by Histopaque-1077density gradient centrifugation (Sigma Diagnostics, St. Louis, Mo.).Cells were washed twice with Iscove's Modified Dulbecco's Medium (IMDM)supplemented with 10% heat-inactivated fetal bovine serum (FBS), andresuspended in 5 ml of ice-cold IMDM/10% FBS. Viable cells were countedby staining with trypan blue. Cells were mixed with an equal volume of85% FBS/15% DMSO and frozen in 1 ml aliquots for storage in liquidnitrogen.

Immunophenotyping showed that >90% of the CD45+ lymphocyte populationexpressed IgD, kappa light chain, CD5, CD19, and CD23. This populationalso expressed low levels of IgM and CD20. Approximately 50% of thecells expressed high levels of CD38. The cells were negative for lambdalight chain, CD10 and CD138.

An aliquot of the cells was thawed, washed, and resuspended at a densityof 10⁷/mL in IMDM supplemented with 20% heat-inactivated FBS, 2 mML-glutamine, 100 units/ml penicillin, 100 μg/ml streptomycin, 50 μM2-mercaptoethanol, and 5 ng/ml recombinant human IL-4 (R & D Systems,Minneapolis, Minn.). The cells were cultured at 37° C. in a humidified5% CO₂ atmosphere. The medium was partially replaced every 4 days untilsteady growth was observed. After 5 weeks, the number of cells in theculture began to double approximately every 4 days. This cell line wasdesignated CLL-AAT.

Characterization of the Cell Line

Immunophenotyping of the cell line by flow cytometry showed highexpression of IgM, kappa light chain, CD23, CD38, and CD138, moderateexpression of CD19 and CD20, and weak expression of IgD and CD5. Thecell line was negative for lambda light chain, CD4, CD8, and CD10.

Immunophenotyping of the cell line was also done by whole cell ELISAusing a panel of rabbit scFv antibodies that had been selected forspecific binding to primary B-CLL cells. All of these CLL-specific scFvantibodies also recognized the MT-CLL cell line. In contrast, themajority of the scFvs did not bind to two cell lines derived from B celllymphomas: Ramos, a Burkitt's lymphoma cell line, and RL, anon-Hodgkin's lymphoma cell line.

EXAMPLE 2

Selection of scFv Antibodies for B-CLL-Specific Cell Surface AntigensUsing Antibody Phage Display and Cell Surface Panning

Immunizations and scFv Antibody Library Construction

Peripheral blood mononuclear cells (PBMC) were isolated from blood drawnfrom CLL patients at the Scripps Clinic (La Jolla, Calif.). Two rabbitswere immunized with 2×10⁷ PBMC pooled from 10 different donors with CLL.Three immunizations, two sub-cutaneous injections followed by oneintravenous injection, were done at three week intervals. Serum titerswere checked by measuring binding of serum IgG to primary CLL cellsusing flow cytometry. Five days after the final immunization, spleen,bone marrow, and PBMC were harvested from the animals. Total RNA wasisolated from these tissues using Tri-Reagent (Molecular ResearchCenter, Inc). Single-chain Fv (scFv) antibody phage display librarieswere constructed as previously described (Barbas et al., (2001) PhageDisplay: A Laboratory Manual, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y.). For cell surface panning, phagemid particles fromthe reamplified library were precipitated with polyethylene glycol(PEG), resuspended in phosphate-buffered saline (PBS) containing 1%bovine serum albumin (BSA), and dialysed overnight against PBS.

Antibody Selection by Cell Surface Panning

The libraries were enriched for CLL cell surface-specific antibodies bypositive-negative selection with a magnetically-activated cell sorter(MACS) as described by Siegel et al. (1997, J. Immunol. Methods206:73-85). Briefly, phagemid particles from the scFv antibody librarywere preincubated in MPBS (2% nonfat dry milk, 0.02% sodium azide inPBS, pH 7.4) for 1 hour at 25° C. to block nonspecific binding sites.Approximately 10⁷ primary CLL cells were labeled with mouse anti-CD5 IgGand mouse anti-CD19 IgG conjugated to paramagnetic microbeads (MiltenyiBiotec, Sunnyvale, Calif.). Unbound microbeads were removed by washing.The labeled CLL cells (“target cells”) were mixed with an excess of“antigen-negative absorber cells”, pelleted, and resuspended in 50 μl(10¹⁰-10¹¹ cfu) of phage particles. The absorber cells serve to soak upphage that stick non-specifically to cell surfaces as well as phagespecific for “common” antigens present on both the target and absorbercells. The absorber cells used were either TF-1 cells (a humanerythroleukemia cell line) or normal human B cells isolated fromperipheral blood by immunomagnetic negative selection (StemSep system,StemCell Technologies, Vancouver, Canada). The ratio of absorber cellsto target cells was approximately 10 fold by volume. After a 30 minuteincubation at 25° C., the cell/phage mixture was transferred to aMiniMACS MS⁺ separation column. The column was washed twice with 0.5 mlof MPBS, and once with 0.5 ml of PBS to remove the unbound phage andabsorber cells. The target cells were eluted from the column in 1 ml ofPBS and pelleted in a microcentrifuge at maximum speed for 15 seconds.The captured phage particles were eluted by resuspending the targetcells in 200 μl of acid elution buffer (0.1 N HCl, pH adjusted to 2.2with glycine, plus 1 μg/ml BSA). After a 10 minute incubation at 25° C.,the buffer was neutralized with 12 μL of 2M Tris base, pH10.5, and theeluted phage were amplified in E. coli for the next round of panning.For each round of panning, the input and output phage titers weredetermined. The input titer is the number of reamplified phage particlesadded to the target cell/absorber cell mixture and the output titer isthe number of captured phage eluted from the target cells. An enrichmentfactor (E) is calculated using the formula E=(R_(n) output/R_(n)input)/(R₁ output/R₁ input). In most cases, an enrichment factor of10²-10³ fold should be attained by the third or fourth round.

Analysis of Enriched Antibody Pools Following Panning

After 3-5 rounds of panning, the pools of captured phage were assayedfor binding to CLL cells by flow cytometry and/or whole cell ELISA:

-   1. To produce an entire pool in the form of HA-tagged soluble    antibodies, 2 ml of a non-suppressor strain of E. coli (e.g.    TOP10F′) was infected with 1 μl (10⁹-10¹⁰ cfu) of phagemid    particles. The original, unpanned library was used as a negative    control. Carbenicillin was added to a final concentration of 20 μM    and the culture was incubated at 37° C. with shaking at 250 rpm for    1 hour. Eight ml of SB medium containing 50 μg/ml carbenicillin was    added and the culture was grown to an OD 600 of ˜0.8. IPTG was added    to a final concentration of 1 mM to induce scFv expression from the    Lac promoter and shaking at 37° C. was continued for 4 hours. The    culture was centrifuged at 3000×g for 15′. The supernatant    containing the soluble antibodies was filtered and stored in 1 ml    aliquots at −20° C.-   2. Binding of the scFv antibody pools to target cells vs. absorber    cells was determined by flow cytometry using high-affinity Rat    Anti-HA (clone 3F10, Roche Molecular Biochemicals) as secondary    antibody and PE-conjugated Donkey Anti-Rat as tertiary antibody.-   3. Binding of the antibody pools to target cells vs. absorber cells    was also determined by whole-cell ELISA as described below.    Screening Individual scFv Clones Following Panning

To screen individual scFv clones following panning, TOP10F′ cells wereinfected with phage pools as described above, spread onto LB platescontaining carbenicillin and tetracycline, and incubated overnight at37° C. Individual colonies were inoculated into deep 96-well platescontaining 0.6-1.0 ml of SB-carbenicillin medium per well. The cultureswere grown for 6-8 hours in a HiGro shaking incubator (GeneMachines, SanCarlos, Calif.) at 520 rpm and 37° C. At this point, a 90 μl aliquotfrom each well was transferred to a deep 96-well plate containing 10 μLof DMSO. This replica plate was stored at −80 C. IPTG was added to theoriginal plate to a final concentration of 1 mM and shaking wascontinued for 3 hours. The plates were centrifuged at 3000×g for 15minutes. The supernatants containing soluble scFv antibodies weretransferred to another deep 96-well plate and stored at −20° C.

A sensitive whole-cell ELISA method for screening HA-tagged scFvantibodies was developed:

-   1. An ELISA plate is coated with concanavalin A (10 mg/ml in 0.1 M    NaHCO₃, pH8.6, 0.1 mM CaCl₂).-   2. After washing the plate with PBS, 0.5-1×10⁵ target cells or    absorber cells in 50 μl of PBS are added to each well, and the plate    is centrifuged at 250×g for 10 minutes.-   3. 50 μl of 0.02% glutaraldehyde in PBS are added and the cells are    fixed overnight at 4° C.-   4. After washing with PBS, non-specific binding sites are blocked    with PBS containing 4% non-fat dry milk for 3 hours at room    temperature.-   5. The cells are incubated with 50 μl of soluble, HA-tagged scFv or    Fab antibody (TOP10F′ supernatant) for 2 hours at room temperature,    then washed six times with PBS.-   6. Bound antibodies are detected using a Mouse Anti-HA secondary    antibody (clone 12CA5) and an alkaline phosphatase (AP)-conjugated    Anti-Mouse IgG tertiary antibody. Color is developed with the    alkaline phosphatase substrate PNPP and measured at 405 nm using a    microplate reader.

Primary screening of the scFv clones was done by ELISA on primary CLLcells versus normal human PBMC. Clones which were positive on CLL cellsand negative on normal PBMC were rescreened by ELISA on normal human Bcells, human B cell lines, TF-1 cells, and the CLL-MT cell line. Theclones were also rescreened by ELISA on CLL cells isolated from threedifferent patients to eliminate clones that recognized patient-specificor blood type-specific antigens. Results from representative ELISAs areshown in FIGS. 2-6 and summarized in Table 1.

The number of unique scFv antibody clones obtained was determined by DNAfingerprinting and sequencing. The scFv DNA inserts were amplified fromthe plasmids by PCR and digested with the restriction enzyme BstNI. Theresulting fragments were separated on a 4% agarose gel and stained withethidium bromide. Clones with different restriction fragment patternsmust have different amino acid sequences. Clones with identical patternsprobably have similar or identical sequences. Clones with unique BstNIfingerprints were further analyzed by DNA sequencing. Twenty-fivedifferent sequences were found, which could be clustered into 16 groupsof antibodies with closely related complementarity determining regions(Table 1).

Characterization of scFv Antibodies by Flow Cytometry

The binding specificities of several scFv antibodies were analyzed by3-color flow cytometry. PBMC isolated from normal donors were stainedwith FITC-conjugated anti-CD5 and PerCP-conjugated anti-CD19. Stainingwith scFv antibody was done using biotin-conjugated anti-HA as secondaryantibody and PE-conjugated streptavidin. Three antibodies, scFv-2,scFv-3, and scFv-6, were found to specifically recognize the CD19⁺ Blymphocyte population (data not shown). The fourth antibody, scFv-9,recognized two distinct cell populations: the CD19⁺ B lymphocytes and asubset of CD5⁺ T lymphocytes (See, FIG. 7). Further characterization ofthe T cell subset showed that it was a subpopulation of the CD4⁺ CD8⁻T_(H) cells.

To determine if the antigens recognized by the scFv antibodies wereoverexpressed on primary CLL cells, PBMC from five CLL patients and fivenormal donors were stained with scFv and compared by flow cytometry(FIG. 8 and Table 2). By comparing the mean fluorescent intensities ofthe positive cell populations, the relative expression level of anantigen on CLL cells vs. normal cells could be determined. One antibody,scFv-2, consistently stained CLL cells less intensely than normal PBMC,whereas scFv-3 and scFv-6 both consistently stained CLL cells morebrightly than normal PBMC. The fourth antibody, scFv-9, stained two ofthe five CLL samples much more intensely than normal PBMC, but gave onlymoderately brighter staining for the other three CLL samples (FIG. 8 andTable 2). As seen in FIG. 8., the antigens recognized by scFv-3 andscFv-9 are overexpressed on the primary CLL tumor from which the CLL-AATcell line was derived. Primary PBMC from the CLL patient used toestablish the CLL-MT cell line or PBMC from a normal donor were stainedwith scFv antibody and analyzed by flow cytometry. ScFv-3 and scFv-9stain the CLL cells more brightly than the normal PBMC as measured bythe mean fluorescent intensities.

This indicates that the antigens for scFv-3 and scFv-6 are overexpressedapproximately 2-fold on most if not all CLL tumors, whereas scFv-9 isoverexpressed 3 to 6-fold on a subset of CLL tumors.

Identification of Antigens Recognized by scFv Antibodies byImmunoprecipitation (IP) and Mass Spectrometry (MS)

To identify the antigens for these antibodies, scFvs were used toimmunoprecipitate the antigens from whole cell lysates or lysatesprepared from microsomal fractions of cells. The immunoprecipitatedantigens were purified by SDS-PAGE and identified by MALDI-MS analysis(data not shown). ScFv-2 immunoprecipitated a 110 kd antigen from bothRL and CLL-AAT cells. This was identified as the B cell-specific markerCD19. ScFv-3 and scFv-6 both immunoprecipitated a 45 kd antigen fromCLL-AAT cells. This was identified as CD23, which is a known marker forCLL and activated B cells. ScFv-9 immunoprecipitated a 50 kd antigenfrom CLL-MT cells. However, we have not yet isolated this antigen insufficient quantities for MALDI-MS analysis, because its expressionappears to have been downregulated on the CLL-AAT cell line.

REFERENCES

The following references are incorporated herein by reference to morefully describe the state of the art to which the present inventionpertains. Any inconsistency between these publications below or thoseincorporated by reference above and the present disclosure shall beresolved in favor of the present disclosure.

-   Almasri, N M et al. (1992). Am J Hemato 140 259-263.-   10 Hainsworth, JD (2000). Oncologist 2000;5(5):376-84-   Nilsson, K (1992). Burn Cell. 5(1):25-41.-   Pu, QQ and Bezwoda, W (2000). Anticancer Res. 20(4):2569-78.-   Walls A Vet al. (1989). Int. J. Cancer 44846-853.

It will be understood that various modifications may be made to theembodiments disclosed herein. Those skilled in the art will envisionother modifications within the scope of the claims appended hereto.

1. The cell line CLL-AAT, deposited under ATCC Accession No. ______. 2.A method for preparing an antibody, comprising the steps of: (i)generating an antibody to at least one antigen presented on the surfaceof a CLL cell line according to claim 1; and (ii) determining that saidantibody binds with an antigen associated with CLL cells.
 3. A method asin claim 2 wherein the step of determining comprises determining thatsaid antibody binds with an antigen that is upregulated on CLL cells. 4.A method as in claim 2 wherein the step of determining comprisesdetermining that said antibody binds with an antigen specific for CLLcells.
 5. A method according to claim 2, wherein the antibody isgenerated by immunization of an organism with a CLL cell line cell, orpart thereof, according to claim
 1. 6. A method according to claim 5,wherein the antibody is generated by panning a synthetic antibodylibrary with a CLL cell line according to claim
 1. 7. A method accordingto claim 6, wherein the library is screened by phage display to isolatethe antibody.
 8. A method for characterizing an antibody that binds toCLL cells, comprising assessing the binding of the antibody to a cellline according to claim
 1. 9. A method according to claim 8, wherein theantibody is isolated by panning an antibody library with primary CLLcells isolated from one or more a patients suffering from CLL.
 10. Anantibody produced by the method of claim
 2. 11. An antibody that bondsto an antigen that is upregulated by the cell line of claim
 1. 12. Anantibody having a sequence selected from the group consisting of SEQ.ID. NO: 1, SEQ. ID. NO: 2, SEQ. ID. NO: 3, SEQ. ID. NO: 4, SEQ. ID. NO:5, SEQ. ID. NO: 6, SEQ. ID. NO: 7, SEQ. ID. NO: 8, SEQ. ID. NO: 9, SEQ.ID. NO: 10, SEQ. ID. NO: 11, SEQ. ID. NO: 12, SEQ. ID. NO: 13, SEQ. ID.NO: 14, SEQ. ID. NO: 15, SEQ. ID. NO: 16, SEQ. ID. NO: 17, SEQ. ID. NO:18, SEQ. ID. NO: 19, SEQ. ID. NO: 20, SEQ. ID. NO: 21, SEQ. ID. NO: 22,SEQ. ID. NO: 23, SEQ. ID. NO: 24, and SEQ. ID. NO: 25.